Nell-1 enhanced bone mineralization

Abstract

This invention pertains to the discovery that the human NELL-1 gene induces or upregulates bone mineralization. The HELL-1 gene or gene product thus provides a convenient target for screening for modulators of bone mineralization. In addition, HELL-1 can be used to facilitate repair of bone fractures and/or to generally increase bone density.

1 . A method of modulating calvarial osteoblast differentiation and mineralization in a human being, said method comprising: 
 altering expression or activity of Nell-1 in the human being, wherein increased expression or activity of Nell-1 increases osteoblast differentiation or mineralization and decreased expression or activity of Nell-1 decreases osteoblast differentiation or mineralization in the human being.    
     
     
         2 . The method of  claim 1 , wherein Nell-1 expression or activity is inhibited by a method selected from the group consisting of an anti-Nell-1 antisense molecule, a Nell-1 specific riibozyme, a Nell-1 specific catalytic DNA, a Nell-1 specific RNAi, anti-Nell-1 intrabodies, and gene therapy approaches that knock out Nell-1 in target cells and/or tissues.  
     
     
         3 . The method of  claim 1 , wherein Nell-1 expression or activity is increased by a method selected from the group consisting of transfecting a cell with an exogenous nucleic acid expressing Nell-1, and transfecting a cell with a Nell-1 protein.  
     
     
         4 . The method of  claim 2 , wherein said Nell-1 expression or activity is inhibited in the human, and 
 wherein the human being is experiencing abnormal cranial suture development.    
     
     
         5 . The method of  claim 4 , wherein said abnormal cranial suture development comprises craniosynostosis (CS).  
     
     
         6 . A method of facilitating latent TGF-β1 activation in a human being, said method comprising administering exogenous Nell-1 to said human being, or increasing expression activity of endogenous Nell-1 in the human being.  
     
     
         7 . A method of activating or sequestering a member of the TGF-β superfamily in a human being, said method comprising administering exogenous Nell-1 to said human being, or increasing expression activity of endogenous Nell-1 in the human being.  
     
     
         8 . A method of screening for an agent that modulates osteoblast differentiation, said method comprising: 
 contacting a test cell which is a human cell containing a Nell-1 gene with a test agent; and    detecting a change in the expression level of a Nell-1 gene or the activity of Nell-1 in said test cell as compared to the expression of the Nell-1 gene or the activity of Nell-1 in a control cell where a difference in the expression level of Nell-1 or the activity of Nell-1 in the test cell and the control cell indicates that said agent modulates bone mineralization.    
     
     
         9 . The method of  claim 8 , wherein said control is a negative control cell contacted with said test agent at a lower concentration than said test cell.  
     
     
         10 . The method of  claim 9 , wherein said lower concentration is the absence of said test agent.  
     
     
         11 . The method of  claim 8 , wherein said control is a positive control cell contacted with said test agent at a higher concentration than said test cell.  
     
     
         12 . The method of  claim 8 , further comprising recording test agents that alter expression of the Nell-1 nucleic acid or the Nell-1 protein in a database of modulators of Nell-1 activity or in a database of modulators of bone mineralization.  
     
     
         13 . The method of  claim 8 , wherein the expression level of Nell-1 is detected by measuring the level of Nell-1 mRNA in said cell.  
     
     
         14 . The method of  claim 13 , wherein said level of Nell-1 mRNA is measured by hybridizing said mRNA to a probe that specifically hybridizes to a Nell-1 nucleic acid.  
     
     
         15 . The method of  claim 14 , wherein said hybridizing is according to a method selected from the group consisting of a Northern blot, a Southern blot using DNA derived from the Nell-1 RNA, an array hybridization, an affinity chromatography, and an in situ hybridization.  
     
     
         16 . The method of  claim 15 , wherein said probe is a member of a plurality of probes that forms an array of probes.  
     
     
         17 . The method of  claim 13 , wherein said level of Nell-1 mRNA is measured using a nucleic acid amplification reaction.  
     
     
         18 . The method of  claim 8 , wherein said level of Nell-1 is detected by determining the expression level of a Nell-1 protein in said biological sample.  
     
     
         19 . The method of  claim 18 , wherein said detecting is via a method selected from the group consisting of capillary electrophoresis, a Western blot, mass spectroscopy, ELISA, immunochromatography, and immunohistochemistry.  
     
     
         20 . The method of  claim 8 , wherein said cell is cultured ex vivo.  
     
     
         21 . The method of  claim 8 , wherein said test agent is not an antibody.  
     
     
         22 . The method of  claim 8 , wherein said test agent is not a protein.  
     
     
         23 . A method of altering Nell-1 expression in a human cell, said method comprising altering the expression or activity of Msx2 and/or Cafa1 in the human cell.  
     
     
         24 . The method of  claim 23 , comprising upregulating Cbfa1 expression or activity in the human cell to upregulate Nell-1 expression or activity.  
     
     
         25 . The method of  claim 23 , comprising upregulating Msx2 expression or activity in the human cell to downregulate Nell-1 expression or activity.  
     
     
         26 . A method of screening for an agent that modulates Nell-1 expression or activity in a human being, said method comprising: 
 contacting a test cell which is a human cell containing a Cbfa1 and/or an Msx2 gene with a test agent; and    detecting a change in the expression level of an Cbfa1 and/or an Msx2 gene or the activity of Cbfa1 and/or an Msx2 in said test cell as compared to the expression of the Cbfa1 and/or an Msx2 gene or the activity of Cbfa1 and/or an Msx2 in a control cell where a difference in the expression level of Cbfa1 and/or an Msx2 or the activity of Cbfa1 and/or an Msx2 in the test cell and the control cell indicates that said agent modulates Nell-1 expression or activity.    
     
     
         27 . The method of  claim 26 , wherein said control is a negative control cell contacted with said test agent at a lower concentration than said test cell.  
     
     
         28 . The method of  claim 27 , wherein said lower concentration is the absence of said test agent.  
     
     
         29 . The method of  claim 26 , wherein said control is a positive control cell contacted with said test agent at a higher concentration than said test cell.  
     
     
         30 . The method of  claim 26 , further comprising recording test agents that alter expression of Cbfa1 and/or an Msx2 gene or the activity of Cbfa1 and/or an Msx2 in a database of modulators of Nell-1 activity or in a database of modulators of bone mineralization.  
     
     
         31 . The method of  claim 26 , wherein the expression level of Nell-1 is detected by measuring the level of Cbfa1 and/or an Msx2 mRNA in said cell.  
     
     
         32 . The method of  claim 31 , wherein said level of Cbfa1 and/or an Msx2 mRNA is measured by hybridizing said mRNA to a probe that specifically hybridizes to a Cbfa1 and/or an Msx2 nucleic acid.  
     
     
         33 . The method of  claim 32 , wherein said hybridizing is according to a method selected from the group consisting of a Northern blot, a Southern blot using DNA derived from the Cbfa1 and/or Msx2 RNA, an array hybridization, an affinity chromatography, and an in situ hybridization.  
     
     
         34 . The method of  claim 33 , wherein said probe is a member of a plurality of probes that forms an array of probes.  
     
     
         35 . The method of  claim 31 , wherein said level of Cbfa1 and/or Msx2 RNA is measured using a nucleic acid amplification reaction.  
     
     
         36 . The method of  claim 26 , wherein said level of Cbfa1 and/or Msx2 is detected by determining the expression level of a Cbfa1 and/or Msx2 protein in said biological sample.  
     
     
         37 . The method of  claim 36 , wherein said detecting is via a method selected from the group consisting of capillary electrophoresis, a Western blot, mass spectroscopy, ELISA, immunochromatography, and immunohistochemistry.  
     
     
         38 . The method of  claim 26 , wherein said cell is cultured ex vivo.  
     
     
         39 . The method of  claim 26 , wherein said test agent is not an antibody.  
     
     
         40 . The method of  claim 26 , wherein said test agent is not a protein.  
     
     
         41 . A pharmaceutical formulation comprising: 
 one or more active agents in an amount effective for increasing osteoblast differentiation or mineralization in a human being selected from the group consisting of a nucleic acid encoding a Nell-1 protein, a Nell-1 protein, and an agent that alters expression or activity of a Nell-1 protein; and 
 a pharmaceutically acceptable excipient.